Zeolite imidazate framework material and preparation method and application of hydrogen production zeolite imidazole nano-carrier

Abstract

The invention discloses a preparation method and application of a zeolite imidazate framework material and a hydrogen-producing zeolite imidazole nano-carrier, and the preparation method comprises the following steps: dissolving zinc nitrate hexahydrate and cetyltrimethylammonium bromide in deionized water to form a solution A, dissolving 2-methylimidazole in deionized water to form a solution B, finally, dropwise adding the solution A into the solution B, and stirring for reaction to obtain the zeolite imidazate framework material. Dropwise adding a sodium borohydride aqueous solution into the zeolite imidazate framework material, stirring and reacting to obtain a solution C; and centrifuging the solution C by using an ultrafiltration tube, and washing by using deionized water to obtain the hydrogen-producing zeolite imidazole nano-carrier. The small-size reductive ZIF-8 is simple in preparation process and does not need complex hydrogen production triggering conditions, so that the small-size reductive ZIF-8 can efficiently and controllably produce hydrogen in a tumor, and tumor cell death is effectively stimulated and a tumor immune microenvironment is activated by inhibiting mitochondrial respiration and destroying a redox steady state in tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of hydrogen therapy for tumor immunotherapy, and in particular to a preparation method and application of a zeolite imidazolate skeleton material and a hydrogen-producing zeolite imidazole nanocarrier. Background Art

[0002] In recent years, hydrogen therapy has gradually emerged as an innovative tumor treatment method in scientific research and clinical trials. Hydrogen has excellent selective antioxidant, cell signal regulation and energy metabolism regulation effects, which can not only effectively inhibit the proliferation and spread of tumor cells, but also significantly reduce the adverse reactions caused by radiotherapy and chemotherapy.

[0003] Currently, commonly used hydrogen delivery methods include direct hydrogen delivery, hydrogen delivery via delivery platforms, and in situ hydrogen production inside tumors. Among them, direct hydrogen delivery is limited in therapeutic effect due to low solubility and uncontrollable targeted delivery and release. The hydrogen delivery platform based on metal hydrides can increase the gas loading capacity while achieving intra-tissue release. However, the high preparation difficulty, high water reactivity and low biosafety of metal hydrides limit their application in in vivo delivery. The hydrogen production strategy has the advantages of controllable, continuous, targeted release and low biotoxicity in in situ hydrogen production in tumors, but the complex catalytic materials and triggering conditions limit its application. Therefore, the development of hydrogen production carriers that are simple to prepare and have good biocompatibility is still a major research direction.

[0004] Metal-organic frameworks (MOFs) are lattice structure materials formed by the coordination of metal ions or clusters with organic ligands. They have the advantages of adjustable chemical composition, modifiable structure, and biodegradability. They are widely used in gas adsorption and separation, catalysis, drug delivery and other fields.

[0005] Zeolitic imidazolate frameworks (ZIFs) are a new type of composite material that combines the advantages of MOFs and zeolites. They have highly ordered pore structures and stable physical properties similar to zeolites, and they also have the characteristics of MOFs in terms of chemical structure diversity and controllability. This makes ZIFs not only perform well in adsorption separation and catalytic reactions, but also show potential in emerging application areas such as drug delivery and gas storage.

[0006] In terms of drug delivery, the controllable pore size and surface properties of ZIFs make them ideal drug carriers. By precisely regulating their pore structure, the release rate and efficiency of drugs can be effectively controlled, thereby improving the therapeutic effect and reducing the side effects of drugs. This property is particularly important for various types of drugs because they can maintain stability during delivery and ensure that the drug reaches the effective concentration of the target tissue or cell in the body. The chemical stability of ZIFs ensures the stability of drug carriers under various environmental conditions, including different pH values ​​and temperature changes that may be encountered during delivery. This stability not only helps the long-term storage of drugs, but also ensures the consistency and expected effect of drug release in the body. Therefore, as part of the drug delivery system, ZIFs not only expand the possibilities of drug treatment, but also improve the safety and effectiveness of treatment options, bringing new hope and development opportunities to the fields of medicine and biopharmaceuticals.

[0007] In the field of gas storage and separation, ZIFs have shown significant advantages due to their highly customized pore structures, especially in CO2 capture and H2 storage. These capabilities are of great significance for addressing global warming and promoting the widespread use of renewable energy. First, the pore structure of ZIFs can be designed to match the size and affinity of specific gas molecules, thereby achieving efficient gas adsorption and release. For example, for CO2 capture, the pore size and chemical properties of ZIFs enable them to efficiently adsorb CO2, which helps reduce greenhouse gas emissions in industrial processes. This ability has significant potential for achieving emission reduction targets and addressing climate change. In addition, the application of ZIFs in H2 storage has also attracted much attention. As a clean energy source, H2 has an important position in energy transformation due to its high energy density and zero emission characteristics. ZIFs can safely and efficiently store and release hydrogen through their adjustable pore structure, which helps solve the storage and transportation problems of hydrogen energy and promote the widespread use of renewable energy. Summary of the invention

[0008] The present invention provides a method for preparing a zeolite imidazole ester framework material and a hydrogen-producing zeolite imidazole nanocarrier and its application. The present invention uses safe and non-toxic ZIF-8 nanoparticles as a template to synthesize a hydrogen-producing zeolite imidazole nanocarrier (ReZIF-8) through an aqueous phase reduction reaction. The principle is to use the strong reducing property of sodium borohydride to convert Zn in ZIF-8 into 2+ A part of it is reduced to Zn, which enables it to produce hydrogen under acidic conditions. Its hydrogen production ability is used to change the tumor redox microenvironment to promote immune activation and trigger antigen-specific immune response through immunogenic death.

[0009] A method for preparing a zeolite imidazolate framework material comprises the following steps:

[0010] 1) Dissolve zinc nitrate hexahydrate and hexadecyltrimethylammonium bromide in deionized water to form solution A, dissolve 2-methylimidazole in deionized water to form solution B, and finally slowly drop solution A into solution B and stir to react to obtain a zeolite imidazole ester framework material.

[0011] In step 1), the concentration of zinc nitrate hexahydrate in the A solution is 0.01-0.04 g / mL, the concentration of hexadecyltrimethylammonium bromide is 0.33-1.32 mg / mL, the concentration of 2-methylimidazole in the B solution is 0.02-0.08 g / mL, and the volume ratio of solution A to solution B is 1:5-10.

[0012] In step 1), the reaction temperature is 15-35°C.

[0013] In step 1), the reaction is stirred for 12 to 24 hours.

[0014] A method for preparing a hydrogen-producing zeolite imidazole nanocarrier comprises the following steps:

[0015] 1) dissolving zinc nitrate hexahydrate and hexadecyltrimethylammonium bromide in deionized water to form a solution A, dissolving 2-methylimidazole in deionized water to form a solution B, and finally slowly dropping the solution A into the solution B and stirring the reaction to obtain a zeolite imidazole ester framework material;

[0016] 2) dropping the sodium borohydride solution into the zeolite imidazolate framework material and stirring to react to obtain solution C;

[0017] 3) Solution C was centrifuged using an ultrafiltration tube and washed twice with deionized water to obtain a hydrogen-producing zeolite imidazole nanocarrier.

[0018] In step 2), the mass of the sodium borohydride solution is 0.02-0.08 g / mL.

[0019] In step 2), stir for 1 to 4 hours.

[0020] In step 3), the centrifugation temperature is 4°C.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1) Without changing the framework structure of ZIF-8NPs, sodium borohydride is used to directly reduce them into ReZIF-8 with hydrogen-producing properties; ReZIF-8 not only has a simple preparation process, but can also trigger hydrogen production under physiological acidic conditions, which enables it to selectively and efficiently produce hydrogen in the acidic environment inside the tumor and achieve tumor suppression by activating the tumor immune microenvironment.

[0023] 2) Compared with other metal element systems, the hydrogen production system of ReZIF-8 can be evenly dispersed inside the tumor due to its smaller size, excellent dispersibility and stability in solution. In addition, it can quickly generate H2 in the acidic tumor microenvironment, thereby achieving efficient tumor immunotherapy by inducing tumor immunogenic death.

[0024] 3) This small-sized reductive ZIF-8 not only has a simple preparation process, but also does not require complex hydrogen production triggering conditions, which enables it to efficiently and controllably produce hydrogen inside the tumor, and effectively stimulate tumor cell death and activate the tumor immune microenvironment by inhibiting mitochondrial respiration and disrupting the redox homeostasis in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM images of ZIF-8 and ReZIF-8.

[0026] Figure 2 Particle size (a) and Zeta potential (b) of ZIF-8 and ReZIF-8.

[0027] Figure 3 Element distribution of Zn, C, N, and O in ReZIF-8 particles.

[0028] Figure 4 (a) XRD patterns and (b) FT-IR spectra of ZIF-8 and ReZIF-8.

[0029] Figure 5 This is the XPS spectrum of Zn2P of ReZIF-8.

[0030] Figure 6 To detect the gas generated by ReZIF-8 in acidic solution by GC-MS.

[0031] Figure 7 (a) The principle and phenomenon of the color change of ReZIF-8 in the acidic solution of MB-Pt; (b) The time and absorbance curves of ZIF-8NPs and ReZIF-8 measured at an absorption wavelength of 650nm.

[0032] Figure 8 This is a laser confocal image of ReZIF-8 entering B16F10 tumor cells. Blue: cytoplasm; Red: ReZIF-8. Green: cell nucleus;

[0033] Fig. 9 The toxicity test results of ReZIF-8 at different concentrations on B16F10 tumor cells.

[0034] Fig.10The fluorescence quantitative analysis statistical chart of cell death and viability staining of B16F10 tumor cells after treatment with PBS, ZIF-8 and ReZIF-8. The concentration of ReZIF-8 group was 75μg / mL.

[0035] Fig.11 B16F10 tumor cells were cultured in PBS and Zn 2+ , cell apoptosis and necrosis after treatment with ZIF-8 and ReZIF-8.

[0036] Fig.12 Effect of ReZIF-8 on CD80 in RAW264.7 + CD86 + (a) CD40 + (b) Effects of expression.

[0037] Fig.13 Effect of ReZIF-8 on the levels of TNF-α (a) and IL-6 (b) in the culture supernatant of RAW264.7 cells.

[0038] Fig.14 ReZIF-8 is used for anti-tumor in vivo. (a) ReZIF-8 anti-tumor treatment flow chart; (b) Mouse weight growth curve within 19 days of treatment; (c) Mouse tumor volume change curve within 19 days; (d) Mouse survival curve after 31 days of treatment. DETAILED DESCRIPTION

[0039] Example 1

[0040] 1) Synthesis of zeolitic imidazolate framework material (ZIF-8NPs)

[0041] At room temperature 25°C, first dissolve 0.18 g of zinc nitrate hexahydrate and 0.006 g of hexadecyltrimethylammonium bromide in 6 mL of deionized water to form solution A, then dissolve 2.724 g of 2-methylimidazole in 42 mL of deionized water to form solution B, and finally slowly drip solution A into solution B and stir for 12 h.

[0042] 2) Synthesis of hydrogen-producing zeolite imidazole nanocarrier (ReZIF-8)

[0043] At room temperature, 0.5 g of sodium borohydride was first dissolved in 10 mL of deionized water, and then slowly dripped into the formed ZIF-8 solution and stirred continuously for 2 h. Finally, the obtained solution was centrifuged at 4 °C using an ultrafiltration tube to obtain the final product, which was then washed twice with deionized water for later use.

[0044] Characterization of the physicochemical properties of ReZIF-8

[0045] 1) Transmission electron microscopy (TEM) observation

[0046] After diluting the centrifuged ReZIF-8 solution to an appropriate multiple, 10 μL was added to the carbon support film, dried at room temperature, and then observed for morphology and particle size. At the same time, the elemental composition of the particles was analyzed by energy dispersive X-ray spectroscopy (EDS). The acceleration voltage of the instrument was 100 kV.

[0047] 2) Determination of particle size distribution and Zeta potential

[0048] After the ReZIF-8 dispersion dispersed after centrifugation was diluted to an appropriate multiple, the hydrated particle size of the sample was measured by dynamic light scattering (DLS) and the surface potential of the sample was measured by an instrument.

[0049] 3) Fourier transform infrared spectroscopy (FT-IR) analysis

[0050] The freeze-dried sample was ground into powder and then pressed into potassium bromide tablets at 400-4000cm -1 Infrared spectrum test is carried out within the range of

[0051] 4) X-ray powder diffraction (XRD) analysis

[0052] After the freeze-dried samples are fully ground into powder, they are tested on an X-ray diffractometer.

[0053] 5) Gas chromatography (GC) analysis

[0054] The gas generated by the reaction of the sample and the hydrochloric acid (HCl) solution is collected and the gas composition is analyzed by gas chromatography.

[0055] 6) Ultraviolet-visible spectrophotometer (UV-Vis) analysis

[0056] Methylene blue (MB) was prepared into a 100 μg / mL solvent using 0.1M HCl and platinum powder (Pt), and the UV absorption peak of the solution was detected using a UV spectrophotometer. At the same time, a standard curve of solvents of different concentrations and absorbance was established at the same absorption wavelength, and the change in absorbance over time during the reaction between the sample and the solvent was recorded to reflect the hydrogen production capacity of the sample.

[0057] Characterization of biological properties of ReZIF-8

[0058] 1) Evaluation of cellular uptake of ReZIF-8

[0059] First, B16F10 tumor cells with good growth status were cultured at a rate of 1×10 5The cells were inoculated at a density of 1 mL / well on a glass-based culture dish and cultured for 24 hours. The supernatant of the cell culture medium was then removed and the material stained with Cy5.5 (red) was added and incubated for 6 hours. The supernatant was then discarded and the cell nucleus was stained with Hochest33342 (blue) dye and the cytoplasm was stained with CellTrackerTM Green (green) dye according to the instructions. Finally, after washing several times, the endocytosis of ReZIF-8 was observed using a fluorescence microscope.

[0060] 2) Evaluation of the toxicity of ReZIF-8 to B16F10 tumor cells

[0061] First, B16F10 tumor cells with good growth status were cultured at a rate of 1×10 4 The cells were inoculated into a 96-well cell culture plate at a density of 100 μL / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. Then, the 1 mg / mL ReZIF-8 solution was diluted with complete culture medium according to a concentration gradient and added to the 96-well cell culture plate at 100 μL / well. The concentration gradient was set to 0, 3.125, 6.25, 12.5, 25, 50, 75, 100, 150, 200 μg / mL, and 5 samples were repeated in parallel. After continuing to culture for 24 hours, the culture medium was discarded, and 100 μL of 10% enhanced CCK-8 reagent diluted with complete culture medium was added to each well and the cells were incubated for 0.5-1 hour. Finally, the absorbance value of each well was read at 450 nm using an ELISA reader to calculate the cell viability (Viability,%).

[0062] 3) Verification of the type of cell death induced by ReZIF-8 in B16F10 tumor cells

[0063] Apoptosis and necrosis kit was used to verify the killing effect of ReZIF-8 on B16F10 tumor cells: B16F10 tumor cells with good growth status were firstly cultured at 1×10 5 The cells were inoculated into a 24-well cell culture plate at a density of 1 mL / well and cultured for 24 hours. The supernatant of the cell culture medium was then removed and the material was added and incubated for 24 hours. All cells were then collected by digestion with EDTA-free trypsin and stained according to the instructions of the FITC-AnnexinV / PI apoptosis and necrosis kit. Finally, the apoptosis and necrosis of tumor cells after ReZIF-8 treatment were detected by flow cytometry.

[0064] 4) The ability of ReZIF-8 to activate RAW 264.7 cells after treatment of B16F10 tumor cells

[0065] First, B16F10 tumor cells with good growth status were cultured at a rate of 1×10 5The cells were inoculated into a 24-well cell culture plate at a density of 0.5 mL / well and cultured for 24 h. The supernatant of the cell culture medium was removed and the materials were added and incubated for 24 h. RAW264.7 cells with good growth status were inoculated at a density of 1×10 5 The cells were added to the 24-well cell culture plate at a density of 0.5 mL / well and cultured for 24 hours. Finally, the cells were stained with CD80, CD86, CD40 and other dyes, and flow cytometry was used to detect the maturation of RAW 264.7 cells stimulated by ReZIF-8 after B16F10 tumor cells were treated.

[0066] 5) Detection of cytokine levels in the supernatant of ReZIF-8 co-cultured with B16F10 and RAW 264.7 in vitro

[0067] First, the supernatant of the cell culture fluid treated in step 4) was collected and centrifuged at 10000 rpm for 10 min, and then aliquoted into 100 μL / tube and frozen in a -80°C refrigerator. Then, TNF-α, IL-6 and other cytokines were detected according to the instructions of the corresponding kit to verify that ReZIF-8 treated B16F10 tumor cells had an immunomodulatory effect on RAW 264.7.

[0068] Animal experiment verification

[0069] 1) Evaluation of the therapeutic effect of ReZIF-8 on mouse melanoma

[0070] The mice with melanoma (B16F10) animal model were divided into 4 groups, with 5-6 mice in each group. On the 7th, 10th, 13th and 16th days, 100 μL of PBS, Zn 2+ , ZIF-8, and ReZIF-8 (100 μg / piece), among which the Zn 2+ At the same time, starting from the 7th day, the weight, tumor size and survival of all mice were recorded every 3 days until the mice died as determined by animal ethics.

[0071] Results and Discussion

[0072] 1) Characterization of the physicochemical properties of ReZIF-8

[0073] The morphology of ZIF-8 and reduced ZIF-8 (ReZIF-8) nanoparticles was characterized by transmission electron microscopy. Figure 1 As shown, the crystal shapes of ZIF-8 and ReZIF-8 are both approximately square, and their particle sizes are uniform, basically around 150nm.

[0074] Figure 2 The dynamic light scattering (DLS) results in a show that the particle sizes of ZIF-8NPs and ReZIF-8 are similar and have good dispersion. Their size is about 215nm, which is larger than the TEM results, which may be related to the hydration layer. The surface potential of the material ( Figure 2 b) shows that the surface of ReZIF-8 is positively charged, with a magnitude of 24.74mV. Positively charged nanomaterials can enhance their adsorption and internalization on the surface of tumor cells, promote the cellular uptake and release of therapeutic drugs or therapeutic carriers, and thus increase the local concentration and effect of the treatment. Therefore, the positive charge of ReZIF-8 is helpful for the treatment of tumors.

[0075] The presence of elements in the nanoparticles was detected by energy dispersive X-ray spectroscopy (EDS). Figure 3 ). The results show that ReZIF-8 contains elements such as C, N, O, and Zn, which are evenly distributed in the nanoparticles.

[0076] The structure and composition of ReZIF-8 were further analyzed by XRD and FT-IR. Figure 4 a), the peaks in the XRD spectra of the reduced ZIF-8NPs and the unreduced ZIF-8NPs are almost the same, which means that the reduced nanoframe is still ZIF-8, and has the same crystal structure and phase as the unreduced group. Moreover, the characteristic peaks consistent with Standard-ZIF-8 and Standard-ZnO appeared in the XRD spectrum of ReZIF-8, which indicates that ReZIF-8 may be composed of them. Correspondingly, the results of FT-IR ( Figure 4 b) It also confirms that the molecular structure of the material is basically not destroyed during the reduction process. As can be seen from the figure, the spectra of the ReZIF-8 group and the ZIF-8 group are basically the same. -1 The absorption peak of CH stretching vibration can be observed near 1600 cm -1 The absorption peak of C=N can be seen near 421cm, which is due to the vibration of the C=N bond in the Imidazolate ligand. -1 and 530cm -1 The absorption peaks of Zn-N and Zn-O appeared at the surfaces of the samples, which is consistent with the results of EDS and XRD.

[0077] In order to further study the chemical valence state of Zn in ReZIF-8, we conducted XPS test analysis. The results showed two peaks at 1022eV and 1045eV (such as Figure 5), indicating that different chemical environments or chemical states of Zn exist in the sample at the same time. The 1025eV peak usually corresponds to reduced Zn or similar chemical environments, while the 1045eV peak corresponds to oxidized Zn or compounds formed with other elements. Therefore, the presence of these two peaks indicates that there are both reduced forms of Zn and oxidized forms of Zn or compounds formed with other elements in the sample. Therefore, the above results show that some Zn in ReZIF-8 2+ It was successfully reduced to Zn, which also confirmed that Zn-doped ZIF-8 nanoparticles can be prepared by reducing ZIF-8NPs with sodium borohydride.

[0078] When ReZIF-8 is dispersed in an acidic aqueous solution, bubbles can be clearly observed in the aqueous solution, while when it is dispersed in a neutral or alkaline aqueous solution, no bubbles are generated in the aqueous solution. This indicates that ReZIF-8 has pH-responsive degradation capabilities and is therefore responsive to the acidic microenvironment of the tumor. On this basis, GC-MS was used to further identify whether the gas generated is H2. The results are shown in Figure 2. Figure 6 As shown, the retention time of the first peak in the spectrum is 1.68 min, which is consistent with the peak time of the characteristic peak of H2 reported in the literature. The results prove that ReZIF-8 can produce hydrogen under acidic conditions.

[0079] H2 can convert blue methylene blue (MB) into colorless under the catalysis of Pt. Therefore, PBS, ZIF-8 and ReZIF-8 were added to the acidic MB-Pt solution (such as Figure 7 a) We found that the solutions of PBS and ZIF-8 groups were still blue after 10 minutes, and no bubbles were generated in the solutions. However, the solution of ReZIF-8 group quickly changed from blue to colorless, and a large number of bubbles were generated. Using a UV-visible spectrophotometer, the absorbance of the solution at 650nm was analyzed over time ( Figure 7 b) The results show that the concentration of methylene blue in ReZIF-8 decreases over time, while that in ZIF-8 group does not change. The above results further prove that ReZIF-8 can react with acidic solution to generate H2.

[0080] 2) Characterization of biological properties of ReZIF-8

[0081] To verify whether ReZIF-8 can enter tumor cells, we labeled ReZIF-8 with the red fluorescent dye Cy5.5 and marked its nucleus in blue with Hochest 33342 and used CellTracker TM The cytoplasm was labeled green and observed using a confocal laser microscope. Figure 8It shows that red ReZIF-8 is evenly distributed in the cytoplasm of tumor cells, proving that ReZIF-8 can enter tumor cells.

[0082] In order to evaluate the cytotoxicity of ReZIF-8, ReZIF-8 was quantified by freeze-drying and ICP-OES data and then co-cultured with cells for 24 h. The cell activity was determined using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt (CCK-8). Fig. 9 ). It can be seen from the figure that when the concentration of ReZIF-8 is lower than 25μg / mL, it has almost no toxicity to tumor cells, and as the concentration increases, its cytotoxicity becomes stronger and stronger. It is worth noting that when the material concentration reaches 75μg / mL, the subsequent increase in concentration has little effect on cytotoxicity, and the proportion of live cells is about 30% at this time.

[0083] In order to preliminarily evaluate the killing effect of ReZIF-8 on B16F10 tumor cells, we used live / dead cell staining to analyze. Image J was used for fluorescence quantitative analysis, and the statistical results are shown in Fig.10 As shown, the proportion of live cells in the PBS and ZIF-8 groups was as high as about 95%, while the proportion of live cells in the ReZIF-8 group was only about 10%.

[0084] In order to further quantitatively analyze the types of tumor cell death, 2+ We used an apoptosis and necrosis kit to detect the type of cell death after 24 hours of culture of the material and tumor cells. Fig.11 We can see that in the PBS group, about 95% of the cells are still alive, and only a small number of cells are in a state of late apoptosis / death. 2+ It has a small toxic effect on cells. In addition, about 40% of the cells in the ZIF-8 group are still active. However, in the ReZIF-8 group, nearly 80% of the cells are in a late apoptotic state, and there are almost no living cells. Therefore, judging from the results of its flow cytometry, ReZIF-8 can induce apoptosis / necrosis of tumor cells.

[0085] For material treatment of tumors, in order to maintain effective treatment effects for a long time, it is not only necessary to have direct tumor killing ability, but also to enhance the immune response ability of the organism itself. In addition, material-induced cell apoptosis / necrosis will also activate natural immune cells. Therefore, we co-cultured B16F10 tumor cells and RAW264.7 immune cells to simulate the real tumor environment, and analyzed the activation level of ReZIF-8 on its immune system by flow cytometry. The results are as follows Fig.12As shown, with PBS and Zn 2+ Compared with the control group, the addition of ReZIF-8 significantly increased the expression of macrophage CD80 and CD86. At the same time, ReZIF-8 also increased the expression level of CD40, from 0.26% to 78%, an increase of 300 times, indicating that ReZIF-8 plays a role in promoting immune activation, enhancing inflammatory response or improving the effect of immunotherapy.

[0086] At the same time, important antigen-presenting cells in the immune system can release a variety of pro-inflammatory factors, such as TNF-α, IL-6, etc., after sensing pathogens or damage signals. Therefore, we used Elisa kits to detect the changes in cytokines in the culture supernatant of co-cultured cells and found that Zn 2+ There was no significant difference between the ZIF-8 treated group and the PBS treated group, but the levels of TNF-α and IL-6 in the supernatant of the ReZIF-8 treated group were much higher than those of the other control groups ( Fig.13 ), which further proves that ReZIF-8 has the ability to stimulate immune cells.

[0087] 3) Animal experiment verification

[0088] Zinc ion (Zn 2+ ) plays an important role in tumor research. It can regulate multiple cell signaling pathways, inhibit tumor cell proliferation and invasion, and promote tumor cell apoptosis (programmed death). 2+ In order to verify the effect of ReZIF-8-based H2 therapy in inhibiting tumor growth, a melanoma (B16F10) animal model was established using C57BL / 6 female mice and treated with uniform Zn 2+ The PBS group was used as the blank control group, and the Zn 2+ and ZIF-8 groups were the material control group, and ReZIF-8 was the experimental group ( Fig.14 a). From the treatment results, there was no significant difference in the body weight of the four groups of mice during the treatment period ( Fig.14 b), which indicates that ReZIF-8 has no obvious toxic side effects on mice. Fig.14 c shows that Zn 2+ The tumor growth rate of the ZIF-8 group was similar to that of the PBS group, while the tumor growth of the ReZIF-8 group was significantly inhibited and had a significant difference compared with the control group. Fig.14d), all the mice in the three control groups died within 30 days, while the mice in the material group still had a 60% survival rate at 31 days, which indicates that ReZIF-8 can effectively inhibit the growth of melanoma in mice and prolong the survival of mice to a certain extent.

Claims

1. A method for preparing a zeolite imidazolate framework material, characterized in that: The following steps are involved: Zinc nitrate hexahydrate and hexadecyltrimethylammonium bromide are dissolved in deionized water to form a solution A, 2-methylimidazole is dissolved in deionized water to form a solution B, and finally the solution A is dropped into the solution B and stirred to react to obtain a zeolite imidazole ester framework material.

2. The method for preparing the zeolite imidazolate framework material according to claim 1, wherein The concentration of zinc nitrate hexahydrate in the solution A is 0.01-0.04 g / mL, and the concentration of hexadecyltrimethylammonium bromide is 0.33-1.32 mg / mL; The concentration of 2-methylimidazole in the B solution is 0.02-0.08 g / mL; The volume ratio of solution A to solution B is 1:5-10.

3. The method for preparing the zeolite imidazolate framework material according to claim 1, wherein In step 1), the stirring reaction temperature is 15-35°C.

4. The method for preparing the zeolite imidazolate framework material according to claim 1, wherein In step 1), the stirring reaction time is 12 to 24 hours.

5. A method for preparing a hydrogen-producing zeolite imidazole nanocarrier, characterized in that: The following steps are involved: 1) dissolving zinc nitrate hexahydrate and hexadecyltrimethylammonium bromide in deionized water to form a solution A, dissolving 2-methylimidazole in deionized water to form a solution B, and finally dropping the solution A into the solution B and stirring the mixture to react to obtain a zeolite imidazole framework material; 2) dropping a sodium borohydride aqueous solution into the zeolite imidazolate framework material and stirring to react to obtain a solution C; 3) Solution C is centrifuged using an ultrafiltration tube and washed with deionized water to obtain a hydrogen-producing zeolite imidazole nanocarrier.

6. The method for preparing the hydrogen-producing zeolite imidazole nanocarrier according to claim 5, characterized in that: In step 2), the concentration of the sodium borohydride aqueous solution is 0.02 to 0.08 g / mL.

7. The method for preparing the hydrogen-producing zeolite imidazole nanocarrier according to claim 5, characterized in that: In step 2), the stirring reaction time is 1 to 4 hours.

8. The method for preparing the hydrogen-producing zeolite imidazole nanocarrier according to claim 5, characterized in that: In step 3), the centrifugal temperature is 2-6°C.

9. Use of the hydrogen-producing zeolite imidazole nanocarrier prepared according to the preparation method according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

10. Use according to claim 9, characterized in that The anti-tumor drug is a melanoma drug.

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